Cynthia M. Cave

The Cannabinoid Receptor 2 Is Critical for the Host Response to Sepsis

Leukocyte function can be modulated through the cannabinoid receptor 2 (CB2R). Using a cecal ligation and puncture (CLP) model of sepsis, we examined the role of the CB2R during the immune response to an overwhelming infection. CB2R-knock out (KO) mice showed decreased survival as compared with wild-type mice. CB2R-KO mice also had increased serum IL-6 and bacteremia. Twenty-four hours after CLP, the CB2R-deficient mice had increased lung injury. Additionally, CB2R-deficiency led to increased neutrophil recruitment, decreased neutrophil activation, and decreased p38 activity at the site of infection. Consistent with a novel role for CB2R in sepsis, CB2R-agonist treatment in wild-type mice increased the mean survival time in response to CLP. Treatment with CB2R-agonist also decreased serum IL-6 levels, bacteremia, and damage to the lungs compared with vehicle-treated mice. Finally, the CB2R agonist decreased neutrophil recruitment, while increasing neutrophil activation and p38 activity at the site of infection compared with vehicle-treated mice. These data suggest that CB2R is a critical regulator of the immune response to sepsis and may be a novel therapeutic target.

T-cell activation differentially mediates the host response to sepsis.

Survival during sepsis requires both swift control of infectious organisms and tight regulation of the associated inflammatory response. As the role of T cells in sepsis is somewhat controversial, we examined the impact of increasing antigen-dependent activation of CD4 T cells in a murine model of cecal ligation and puncture using T-cell receptor transgenic II (OT-II) mice that are specific for chicken ovalbumin (OVA) in the context of major histocompatibility complex II. Here, we injected OT-II mice with 0, 1, or 100 &mgr;g of OVA and demonstrate that increased antigen treatment resulted in increased numbers of activated splenic CD4 T cells. Vehicle-treated, septic OT-II mice had decreased survival, increased bacterial load, and increased levels of IL-6. Interestingly, this decrease in survival was abrogated when OT-II mice were injected with 1 &mgr;g OVA, which was correlated with normalized bacterial load and levels of IL-6. However, when OT-II mice were injected with 100 &mgr;g OVA, decreased survival was restored but, in contrast to vehicle-treated OT-II mice, had decreased bacterial load and enhanced IL-6 levels. We also observed that neutrophil oxidative burst and phagocytosis were dependent on CD4 T-cell activation. Further, at extreme levels of T-cell activation, intestinal permeability was significantly increased. Altogether, we conclude that too little CD4 T-cell activation produces dysfunctional neutrophils leading to decreased bacteria clearance and survival, whereas too much CD4 T-cell activation produces a neutrophil phenotype that leads to efficient bacterial clearance but with increased tissue damage and mortality.

Alterations in membrane cholesterol cause mobilization of lipid rafts from specific granules and prime human neutrophils for enhanced adherence-dependent oxidant production.

The present study sought to examine the function of membrane lipid rafts in adherence-dependent oxidant production in human neutrophils. Rafts are membrane domains that are rich in glycosphingolipids and cholesterol and are thought to be the foci for formation of signaling complexes in a variety of cells. Disruption of lipid rafts by depletion of membrane cholesterol with the chelating agent methyl-&bgr;-cyclodextrin (M&bgr;CD) has been widely used to examine the function of lipid rafts. Here, we report that treatment of human neutrophils with M&bgr;CD unexpectedly caused priming of these cells, manifested as enhanced adherence-dependent oxidant production. Treatment of neutrophils with M&bgr;CD dose-dependently increased oxidant production after adhesion to fibronectin-coated plates. This priming effect was associated with recruitment of CD11b- and CD66b-rich raft domains from the specific granules, as determined by immunoblot and flow cytometry. Confocal microscopy showed that M&bgr;CD caused otherwise untreated neutrophils to rapidly adhere and spread on fibronectin-coated plates. Furthermore, three-dimensional reconstruction microscopy studies showed that M&bgr;CD caused expansion and coalescence of raft domains that covered most of the cell surface. These large raft domains expressed CD11b primarily in the core of these regions. Our studies demonstrate that cholesterol depletion with M&bgr;CD results in neutrophil priming manifested as enhanced adherence-dependent oxidant production. These studies caution against assumption that any observed M&bgr;CD effects are a function of reduced raft formation.

Formation of focal adhesion-like structures in circulating human neutrophils after severe injury: triggering of a tissue-phase response in the vascular space.

ABSTRACT Neutrophils play a key role in injury to the lung, kidney, liver, and gastrointestinal tract, often seen after major trauma. We evaluated the role of integrin-linked focal adhesions in the primed state, previously identified in peripheral blood neutrophils from severely injured patients. Immunoblot analysis of Triton-insoluble cell fractions revealed that total paxillin content was unchanged in comparison with that found in neutrophils from healthy volunteers, but phosphorylation of paxillin on tyrosine residue 118 was increased by more than 2-fold. Immunoprecipitation with antipaxillin and immunoblotting for proline-rich tyrosine kinase 2 (Pyk2) and for fgr showed significantly more colocalization. Densitometric analysis of total phosphotyrosine profiles also demonstrated significantly more in patient cells as compared with healthy cells. When allowed to adhere to fibronectin-coated plates, healthy and patient cells demonstrate a significant increase in tyrosine phosphorylation from that found in suspension-phase cells. Differential interference contrast microscopy of healthy neutrophils adherent to fibronectin matrices demonstrated rounded cells, without evidence of spreading; spreading was induced by addition of TNF-&agr;. Patient neutrophils spread spontaneously, a response not further enhanced by TNF-&agr;. Confocal imaging using anti-Pyk2 demonstrated aggregation of Pyk2 into punctate structures in patient but not in healthy cells. We conclude that neutrophils from severely injured patients are in a primed state, characterized by formation of focal adhesion-like structures. The identification of such structures in a clinical disease setting where they likely participate in unwanted consequences provides a novel area for study of regulation of neutrophil function.